We want to investigate the molecular processes regulating DNA replication during the animal cell growth cycle. Intact cell studies have shown that replication is restricted to the S period of the cell cycle, and that different controls are exerted in G1, S and G2/M periods. However, intact cells are impermeable to many molecules that could act as regulators, and it is not possible to characterize the processes regulating replication at the molecular level using intact cells. We propose to use subcellular preparations, including permeable cells, to investigate the molecular processes regulating replication. We have developed methods for permeabilizing cultured mammalian cells to small and large molecules and have established conditions in which deoxyribonucleotides are incorporated into DNA at in vivo rates, resulting in replication of about 20% of the genome. This system allows investigation of the molecular processes regulating replication under relatively physiological conditions. Permeable cells will be prepared from synchronized cultures; and we will investigate the process regulating replication in different periods of the cell cycle by adding or removing the appropriate regulatory molecules. We will characterize cytoplasmic factors and examine their role in regulating replication, investigate the role of cAMP and protein phosphorylation in controlling replication, identify proteins involved in replication, study the processes regulating entry into S, and examine the processes which restrict replication to the S period. Inhibitors of DNA synthesis will be studied not only to characterize the inhibitors, but also to learn about the processes regulating replication. By studying the processes controlling replication in permeable animal cells, we hope to learn more about the control of DNA replication in the animal cell cycle.